The present invention relates to a process for transforming a C3 plant to provide it with a C4 cycle by introducing two or more enzymes which participate in the C4 photosynthetic pathway.
Three types of photosynthetic pathway are known in higher plants, namely, C3, C4 and CAM types. Leaf tissues of plants having a C4 type photosynthetic pathway (hereafter sometimes referred to as C4 plants) comprise mesophyll cells and bundle sheath cells existing around fibro vascular bundles, forming the specific leaf tissue structure called Kranz-type anatomy. C4 plants fix carbon dioxide into a C4 compound by the action of a phosphoenolpyruvate carboxylase (hereafter sometimes referred to as PEPC) located in the cytoplasm of mesophyll cells. The fixed carbon dioxide is released by decarboxylase in bundle sheath cells, which increases the level of carbon dioxide in the vicinity of ribulose-1,5-biphosphate carboxylase/oxygenase (hereafter sometimes referred to as Rubisco) which is the enzyme for the essential carbon dioxide fixation. The metabolite resulting from the decarboxylation in the bundle sheath cells is transferred into mesophyll cells and converted to phosphoenolpyruvate (hereafter sometimes referred to as PEP), a substrate for PEPC, by the action of the pyruvate, orthophosphate dikinase (hereafter sometimes referred to as PPDK) located in mesophyll cells, with a simultaneous consumption of ATP. Namely, the two types of cells in green leaves of C4 plants are functionally differentiated; mesophyll cell is the place of formation of the C4 compounds at the initial carbon fixation as well as the place of re-generation of the PEPC substrate, while the bundle sheath cell is the place of decarboxylation of the C4 compound and essential carbon dioxide fixation by way of the Calvin-Benson cycle.
The three steps i.e., the carbon dioxide fixation by PEPC, the release of carbon dioxide in the vicinity of Rubisco, and the re-generation of PEPC substrate accompanied by the consumption of ATP, constitute a system of cycle reaction which is called a C4 photosynthetic pathway. The pathway provides C4 plants with an enhanced ability to accumulate carbon dioxide, and to avoid the decrease of photosynthetic efficiency which may otherwise take place under high light intensity due to the over production of ATP (avoidance of photoinhibition). These properties are not found in C3 plants having a regular photosynthetic pathway (C3 type photosynthesis). Thus, C4 plants do not exhibit photorespiration as in C3 plants, and therefore, the former shows less deterioration in the efficiency of photo-synthesis than the latter when placed under an atmosphere which is dried, highlight intensity or high temperature. As such, C4 plants are superior to C3 plants in their ability to conduct photosynthesis.
One might expect that a C4 photosynthetic pathway could be introduced in a C3 plant by means of crossing and breeding. However, most species having a C4 photosynthetic pathway and those having a regular C3 photosynthetic pathway are grouped into different genus or family, and crossing between them is difficult. Moreover, an attempt to introduce properties of a C4 plant wherein a C3 plant was crossed with a C4 plant selected from the same genus orache did not succeed (Ohsugi, R. Nogyo-gijutsu (1995) Vol.50, pp.30-36).
Hudspeth, et al. observed that the green leaves of transgenic tobacco into which PEPC gene was introduced under the control of tobacco chlorophyll a/b binding protein gene promoter (cab promoter) showed the doubling or PEPC activity and the increase of malate level (Hudspeth, et al., Plant Physiol., (1992) 98: 458-464). Kogami, et al. observed that the green leaves of transgenic tobacco into which PEPC gene was introduced under the control of cauliflower mosaic virus 35S promoter contained about twice as much PEPC activity as non-transformed toabcco (Kogami, et al., Transgenic Research (1994) Vol.3: 287-296).
Thus, Hudspeth, et al. and Kogami, et al. simply observed the accumulation of the C4 compound malate without confirming any change in photosynthetic property caused by the introduction of PEPC as a single gene into the C3 plant tobacco. The cells of C3 plants are incapable of rapidly decarboxylating a C4 compound to supply the carbon dioxide to the Calvin cycle. Therefore, it would not be possible, by way of a simple introduction of PEPC gene into a C3 plant, in an attempt to provide the plant with the capability of C4 photosynthetic pathway to concentrate carbonate or avoid a photoinhibition, when an improvement in the photosynthetic property of a C3 plant is desired.
Japanese Patent Public Disclosure Hei 8-80197 discloses that a DNA fragment encoding transit peptide was connected with a phosphoenolpyruvate carboxykinase (PCK) gene. The chimeric gene was introduced into rice, which is a C3 plant, whereby the enzyme activity in the crude extract of green leaves was detected as well as the localization of PCK protein in chloroplasts. These facts indicate that it is possible to allow the activity of PCK to localize in chloroplasts. However, no description is made about the establishment of a C4 photosynthetic pathway or change of photosynthetic property in the transgenic plants.
Ichikawa, et al., Nihon Sakumotsu Gakkai Kiji, Vol.63, Suppl.2, (1994), p.247) disclose that when PPDK was introduced into C3 plants, Arabidopsis and tomato, the protein was accumulated in the plants. However, no description is made about the establishment of a C4 photosynthetic pathway or change in the photosynthetic property of the transgenic plants. Japanese Patent Publication Hei 6-12990 discloses the change in the photosynthetic efficiency in the cotyledonous protoplasts of Lycopersicon esculentum wherein carbonic anhydrase (herein under sometimes referred to as CA) protein was incorporated. On the other hand, Majeau et al., disclose that the in vivo over expression of CA did not give rise to any change in the photosynthetic ability of the plant (Plant Mol. Biol. (1994) 25: 337-385).
As discussed above, previous attempts to introduce a gene from a C4 photosynthetic pathway into a C3 plant by a genetic engineering method were limited to the introduction of CA, PEPC, PCK or PPDK gene as a single gene. These attempts failed to confirm any C4 photosynthetic pathway or change in the efficiency of photosynthesis, even if the expression of the introduced gene or the enzyme activity was observed in some attempts.
An object of the present invention is to provide a method for improving the photosynthetic property of C3 plants. Specifically, the present invention provides a method for transforming a C3 plant to provide it with a C4 photosynthetic pathway by means of the introduction of two or more enzymes which take part in C4 photosynthetic pathway.
Another object of the present invention is to provide a plant which has been transformed to have a C4 pathway in accordance with the method of the invention.
A further object of the present invention is to provide a vector which is useful for conducting the transformation of a C3 plant.